Looped, hairpin ribozyme

ABSTRACT

A polyribonucleotide having a thermodynamically stable loop structure and ribozyme activity and a DNA that codes for the polyribonucleotide. The polyribonucleotide has the following structure ##STR1## Direct administration of the ribozyme polyribonucleotide of the present invention into the body of a patient enables a target polyribonucleotide in the body to be efficiently cleaved, thereby allowing it to be used as an anti-AIDS drug.

TECHNICAL FIELD

The present invention relates to a polyribonucleotide having athermodynamically stable loop structure and ribozyme activity, a DNAthat codes for said polyribonucleotide, an expression vector containingsaid DNA, a host cell transfected with said expression vector, and aprocess for cleaving a specific site of a substrate polyribonucleotideby said polyribonucleotide having ribozyme activity.

BACKGROUND ART

The (+) chain of satellite RNA of tobacco ringspot virus and the (+)chain and (-) chain of avocado sunblotch viroid are cleaved by their owncatalytic activity in the presence of Mg²⁺ (Science 231, 1577-1580(1986)). The RNA structure necessary for this cleavage activity has beendetermined and named as Hammerhead ribozyme (Nucleic Acids Res. 14,3627-3640 (1986)). The nucleotide sequences in the vicinity of thecleavage sites of these RNA possesses common sequences, and thesecondary structure of these RNA was predicted from this commonsequences. Uhlenbeck designed a short chain RNA fragment of 19 mer basedon these common sequences, and indicated that said fragmentcatalytically cleaves RNA of 24 mer (Nature, 328, 596-600 (1987)).

In addition, besides viroid and vital satellite RNA, the transcript ofnewt satellite DNA is also reported to have ribozyme nucleotidesequences (Cell, 48, 535-543, (1987)).

The inventors of the present invention chemically synthesized two typesof 21 mer RNA having nucleotide sequences in the vicinity of thecleavage site of this newt satellite DNA transcript. When one of the RNAwas added to the other, a cleavage reaction was found to occur at thesame site as that in nature (FEBS Lett., 228, 228-230, (1988)). Inaddition, based on this result, the inventors of the present inventionfound a process for cleaving other RNA or polyribonucleotide moleculesusing ribozyme (Nucleic Acids Res., 17, 7059-7071, (1989)).

On the other hand, a cleavage reaction was also caused on the (-) chainof the satellite RNA of tobacco ringspot virus, and that cleavage hasbeen determined to occur at a specific site (Nature 323, 349-353(1986)). In addition, the minimum region of RNA necessary for thiscleavage has also been recently clarified (Biochemistry, 28, 4929-4933(1989)). RNA having this catalytic activity is composed of 50nucleotides, and a model having a hairpin loop structure within this RNAhas been advocated. This RNA has been given the name hairpin ribozyme.The present inventors and other research groups converted thenucleotides of this hairpin ribozyme to other nucleotides, and usedthose results to identify several nucleotides that are important in thecleavage reaction (Nature 354, 320-322 (1991), Nucleic Acids Res. 19,6833-6838 (1991)). Further, the group of Burke et al. has recentlyidentified the base sequence which is important in the cleavage reactionand ligation reaction of hairpin ribozyme by in vitro selection methodusing DNA having random variation and PCR (polymerase chain reaction)(Gene & Development 6, 129-134 (1992)). The present inventors havedetermined that catalytic reaction proceeds even for RNA deleting thehairpin loop portion (Nucleic Acids Res. 19, 6833-6838 (1991)).

In addition, progress in RNA synthesis techniques in recent years hasmade it possible to obtain RNA in large volume, and thus, researches onhigher-order structures of RNA and its physicochemical properties areincreasingly made. Bacteriophage T₄ mRNA and E. coli 16S ribosomal RNAcontain 5'CUUCGG3' sequences in high frequency, and it has beendetermined that the hairpin loop structure formed by this sequence isthermodynamically stable (Pro. Natl. Acad. Sci. USA, 85, 1364-1368,(1988): Nature, 346, 680-682, (1990)).

The present inventors prepared a polyribonucleotide wherein thenucleotide sequence of a hairpin loop which exists at one locationwithin the ribozyme is the above-mentioned thermodynamically stable5'CUUCGG3' sequence, and they found that this polyribonucleotidepossesses high ribozyme cleavage activity and accomplished the presentinvention. As a result of having a thermodynamically stable hairpin loopstructure, the ribozyme in the present invention is expected toefficiently cleave target polyribonucleotides in the living body.

SUMMARY OF THE INVENTION

The present invention relates to:

(1) a polyribonucleotide represented by the following general formula(I): ##STR2## wherein, U represents an uracil nucleotide, C a cytosinenucleotide. A an adenine nucleotide, and G a guanine nucleotide.

S represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

V represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

Q₁ through Q_(n) may be the same or different from one another andrepresent either a cytosine nucleotide or guanine nucleotide,

R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n),

W₁ through W_(k) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

X₁ through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, and k, m and n may be the same ordifferent from one another and represent an integer from 1 to 10;

(2) a polyribonucleotide in general formula (I) set forth in (1) whereinW₁ is a cytosine nucleotide or a guanine nucleotide:

(3) a polyribonucleotide represented by the following general formula(II): ##STR3## wherein, represents an uracil nucleotide, C a cytosinenucleotide, A an adenine nucleotide, and G a guanine nucleotide,

S represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

Q₁ through Q_(n) may be the same or different from one another andrepresent either a cytosine nucleotide or guanine nucleotide,

R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n),

W₁ through W_(k) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

X₁ through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, and k, m and n may be the same ordifferent from one another and represent an integer from 1 to 10);

(4) a polyribonucleotide of general formula (II) set forth in (3)wherein W₁ is a cytosine nucleotide or a guanine nucleotide;

(5) a DNA that codes for the polyribonucleotides set forth in (1), (2),(3) or (4);

(6) a recombinant vector that includes the DNA set forth in (5);

(7) a host cell transfected with the recombinant vector set forth in(6);

(8) a process for cleaving polyribonucleotide β at the site indicatedwith the arrow in the formula using polyribonucleotide α in thefollowing general formula (III): ##STR4## wherein, U represents anuracil nucleotide, C a cytosine nucleotide, A an adenine nucleotide, andG a guanine nucleotide,

S represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

V represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

Q₁ through Q_(n) may be the same or different from one another andrepresent either a cytosine nucleotide or guanine nucleotide,

R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n),

W₁ through W_(k) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

X₁ through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

P represents either an uracil nucleotide, adenine nucleotide or cytosinenucleotide,

Y₁ through Y_(k) represent nucleotides respectively complementary to W₁through W_(k),

Z₁ through Z_(m) represent nucleotides respectively complementary to X₁through X_(m), and

k, m and n may be the same or different from one another and representan integer from 1 to 10);

(9) a process for cleaving polyribonucleotide β in general formula (III)set forth in (8), wherein Y₁ is a guanine nucleotide or cytosinenucleotide complementary to W₁, using polyribonucleotide α, wherein W₁is a cytosine nucleotide or guanine nucleotide;

(10) a process for cleaving polyribonucleodide δ at the site indicatedwith an arrow in the formula using polyribonucleotide γ in the followinggeneral formula (IV): ##STR5## wherein, U represents an uracilnucleotide, C a cytosine nucleotide, A an adenine nucleotide, and G aguanine nucleotide,

S represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

Q₁ through Q_(n) may be the same or different from one another andrepresent either a cytosine nucleotide or guanine nucleotide,

R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n),

W₁ through W_(k) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

X₁ through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

P represents either an uracil nucleotide, adenine nucleotide or cytosinenucleotide,

Y₁ through Y_(k) represent nucleotides respectively complementary to W₁through W_(k),

Z₁ through Z_(m) represent nucleotides respectively complementary to X₁through X_(m), and

k, m and n may be the same or different from one another and representan integer from 1 to 10); and

(11) a process for cleaving polyribonucleotide δ in general formula (IV)set forth in (10), wherein Y₁ is a guanine nucleotide or cytosinenucleotide complementary to W₁, using polyribonucleotide γ, wherein W₁is a cytosine nucleotide or guanine nucleotide.

In addition, the present invention also relates to:

(12) a polyribonucleotide comprising in its molecule the nucleotidesequence represented by the following general formula (V): ##STR6##wherein, U represents an uracil nucleotide, C a cytosine nucleotide, Aan adenine nucleotide, and G a guanine nucleotide,

S represents either an adenine nucleotide or cytosine nucleotide,

Q₁ through Q_(n) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n),

W₁ through W₄ may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide cytosinenucleotide or guanine nucleotide,

X₁ through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, and

m and n may be the same or different from each other and represent aninteger from 1 to 10);

(13) a polyribonucleotide in general formula (V) set forth in (12)wherein S is an adenine nucleotide and n is 3;

(14) a process for cleaving polyribonucleotide ζ, which comprising thenucleotide sequence represented by general formula (VI), at the siteindicated with the arrow in the formula using polyribonucleotide ε,which comprising the nucleotide sequence represented by the followinggeneral formula (VI): ##STR7## wherein, U represents an uracilnucleotide, C a cytosine nucleotide, A an adenine nucleotide, and G aguanine nucleotide,

S represents either an adenine nucleotide or a cytosine nucleotide,

Q₁ through Q_(n) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n),

W₁ through W₄ may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

X₁ through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

P represents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide,

L represents either an uracil nucleotide, adenine nucleotide or cytosinenucleotide,

V represents either an adenine nucleotide in the case S is a cytosinenucleotide or an uracil or cytosine nucleotide in the case S is anadenine nucleotide,

Y₁ through Y₄ represent nucleotides respectively complementary to W₁through W₄, and

Z₁ through Z_(m) represent nucleotides respectively complementary to X₁through X_(m),

m and n may be the same or different from each other and represent aninteger from 1 to 10);

(15) a process for cleaving polyribonucleotide ζ in general formula (VI)set forth in (14), wherein L is an uracil nucleotide and V is a cytosinenucleotide, using polyribonucleotide ε, wherein S is an adeninenucleotide and n is 3:

(16) a DNA that codes for the polyribonucleotide set forth in (12) or(13);

(17) a recombinant vector that includes the DNA set forth in (16): and,

(18) a host cell tranfected with the recombinant vector set forth in(17).

The polyribonucleotide of the present invention has a high level ofribozyme activity (to be referred to as a "ribozymepolyribonucleotide"), and has the capacity to specifically cleave apolyribonucleotide having a predetermined sequence in a cell. Thus, inthe case a polyribonucleotide that has a detrimental effect on theliving body is present in a plant, animal or human, saidpolyribonucleotide can be specifically cleaved in the living body byusing this ribozyme polyribonucleotide.

The DNA of the present invention codes for the ribozymepolyribonucleotide of the present invention, and has the capacity tospecifically cleave a desired polyribonucleotide by incorporating itinto a cell using a suitable vector. The target cell may be a plant,animal or human cell.

Preferable examples of the polyribonucleotide of the present inventioninclude the polyribonucleotide set forth in (2), (4) or (13), and thepolyribonucleotide set forth in (4) or (13) are more preferred.

Optimum examples of the polyribonucleotide of the present inventionconsist of the following compounds (b)(SEQ ID NO: 1), (c)(SEQ ID NO: 2),(e)(SEQ ID NO: 4), (f)(SEQ ID NO: 5), (i)(SEQ ID NO: 7), (j)(SEQ ID NO:8), (n)(SEQ ID NO: 9) and (q)(SEQ ID NO: 10). ##STR8##

In addition, the cleavage process of the present invention is preferablythe cleavage process set forth in (9), (11) or (15), with the cleavageprocess set forth in (11) or (15) being more preferred.

The compound of the present invention represented by the above-mentionedgeneral formula (I), (II) or (V) can be used in the form of a salt.Examples of such salts include inorganic or organic salts includingalkaline metals such as sodium and potassium: alkaline earth metals suchas calcium: ammonia: basic amino acids such as lysine and arginine: andalkyl amines such as triethylamine.

The polyribonucleotide of the present invention can be synthesized usinga nucleoside 3'-O-phosphoramidites purchased from American Bionetics,Inc., wherein the 2'-hydroxyl group is protected with atert-butyldimethylsilyl group and the 5'-hydroxyl group is protectedwith a dimethoxytrityl group, with the DNA/RNA automatic synthesizermanufactured by Applied Biosystem, Inc. (Proc. Natl. Acad. Sci. USA, 85,5764-5768, (1988)).

Removal of the β-cyanoethyl group attached to the phosphate acid group,severing of the polyribonucleotide chain from the carrier, and removalof the acyl group of the base portion were carried out by treatment withbase, the protecting group for the 2'-hydroxyl group was removed bytreatment with tetrabutylammonium fluoride, and the protecting group forthe 5'-hydroxyl group was removed by treatment with acid. By performingsubsequent purification with purification procedures normally used inpurification of nucleic acids, examples of which include desalting andvarious types of chromatography such as reverse phase and ion exchangechromatography (including high-performance liquid chromatography), thecompound represented by the above-mentioned general formula (I), (II) or(V) can be obtained.

In vitro cleavage reaction of a substrate polyribonucleotide chain byribozyme polyribonucleotide can be carried out by the proceduredescribed below (JIKKEN IGAKU, 8, 1685-1689, (1990)).

The 5' terminal of the polyribonucleotide used as the substrate islabeled with a radioisotope and so forth. Ribozyme polyribonucleotide isthen added to this labeled polyribonucleotide in a buffer containingmagnesium chloride, followed by warming.

The reaction temperature is preferably 0° to 100° C., and morepreferably 30° to 50° C.

After a predetermined period of time, the reaction is stopped by addingEDTA to the reaction solution, and the solution is subjected tohomochromatography. The cleavage rate can be calculated byquantitatively determining the cleavage products with the Fuji BioimageAnalyzer BAS 2000 System.

A DNA chain that codes for ribozyme polyribonucleotide can besynthesized using the DNA automatic synthesizer manufactured by AppliedBiosystems, Inc. Determination of the sequence of the resulting DNA canbe performed by using, for example, the Maxam-Gilbert Chemicalmodification method (Maxam, A. M. and Gilbert, W. (1980): "Methods inEnzymology" 65, 499-559) or the dideoxynucleotide chain terminationmethod using an M13 phage (Messing, J. and Vieira, J. (1982): Gene 19,269-276).

In addition, this DNA chain can be formed into a double strand chain byannealing, and an expression vector can be constructed by ligating saiddouble strand DNA under the control of a promoter that actsintracellularly using DNA ligase.

The host cell of the present invention can be obtained by introducingthis expression vector into a host cell. In addition, the expressionvector of the present invention can simultaneously be mass produced.

Examples of host cells are as follows.

Examples of host cells of procaryotic cells include Escherichia coli,Bacillus subtilis and so forth. In order to express the target genewithin the host cell, the host cell should be transfected with areplicon originating in a strain able to adapt to the host cell, namelya plasmid vector containing a replication origin and a regulatingsequence. In addition, it is preferable that the vector has a sequencethat is able to give selectivity of transformation (phenotype) to thetransfected cell.

For example, in the case of E. coli, the E. coli K12 strain and so forthare frequently used. Although pBR322 and pUC type plasmids are typicallyused for the vector, the present invention is not limited to the use ofthese, and any known bacterial strains and vectors can be used.

Examples of promoters in the case of E. coli include tryptophan (trp)promoter, lactose (lac) promoter, tryptophan-lactose (tac) promoter,lipoprotein (lpp) promoter, lamda (λ) PL promoters of bacteriophageorigin, and polypeptide chain elongation factor Tu (tufB) promoter andso forth. Any of these promoters can be used for production of theribozyme polyribonucleotide of the present invention.

Although preferable examples of Bacillus subtills include the 207-25strain, while vectors such as pTUB228 (Ohmura K., et al., (1984), J.Biochem., 95, 87-93) are used, the present invention is not limited tothese.

An example of a promoter that is frequently used is the regulatingsequence of the α-amylase gene of Bacillus subtilis.

The cells of vertebrates, insects, yeasts and so forth are included inthe host cells of eucaryotic organisms. Examples of vertebrate cellsthat are frequently used include the mouse cell NIH-3T3 (J. Virol., 4,549-553, (1969)), the monkey cell cos (Gluzman Y., (1981), Cell, 23,175-182), the dihydrofolate reductase-deletion strain of Chinese hamsterovary cells (CHO) (Urlaub, G. and Chasin, L. A. (1980), Proc. Natl.Acad. Sci. USA, 77, 4216-4220) and so forth. The present invention,however, is not limited to these.

Examples of expression vectors of vertebrate cells, that can be usednormally include those having a promoter located upstream from the geneto be expressed, RNA splicing site, polyadenylated site andtranscription terminal sequence and so forth. These may also have areplication origin as necessary. An example of said expression vector ispSV2dhfr and so forth having an SV40 initial promoter (Subramani, S. etal. (1981), Mol. Cell. Biol., 1, 854-864), but the present invention isnot limited to this.

In addition, yeast can also be used as eucaryotes. Examples ofexpression vectors that can be used for eucaryotes such as said yeastsinclude the promoter of the alcohol dehydrogenase gene (Bennetzen, J.and Hall, B. D., (1982), J. Biol. Chem., 257, 3018-3025) and thepromoter of the acid phosphatase gene (Miyanohara, A. et al. (1983),Proc. Natl. Acad. Sci. USA, 80, 1-5) and so forth.

Other procaryotic or eucaryotic host cells can be transformed byintroducing a vector obtained in the manner described above. Moreover,genes can be expressed in the respective host cells by introducing asuitable promoter and sequence involved in transformation into thesevectors.

In the case where E. coli is used as the host cell, for example, vectorsthat can be used as the expression vector are those having a pBR322replication origin, enable autonomous proliferation in E. coli, andwhich are equipped with a transcriptional promoter and translationalstarting signal. Said expression vector can be incorporated in E. coliby the calcium chloride method (Mandel M. and A. Higa, J. Mol. Biol.,53,154, (1970)), the method of Hanahan (Hanahan D. and M. Meselson,Gene, 10, 63, (1980)), electroporation (Neumann E. et al., (1982) EMBOJ., 1, 841-845) and so forth, thus enabling a cell to be obtained thathas been transfected by the desired vector.

Cells transfected with the desired vector obtained as described abovecan be cultured in accordance with conventional methods, and the desiredribozyme polyribonucleotide can be produced within the cells by saidculturing. Various types of media routinely used corresponding to thehost cells being employed can be suitably selected as the medium used insaid culturing. Examples of media that can be used in the case of theabove-mentioned E. coli include tryprone-yeast medium (1.6%Bactotryptone, 1.0% yeast extract and 10.5% NaCl (pH 7.0)) and peptonemedium (Difco) and so forth.

In addition, in the case where the COS cell is used, for example,vectors that can be used as the expression vector are those having anSV40 replication origin, enable autonomous proliferation in COS cellsand which are equipped with a transcriptional promoter, transcriptionalterminating signal and RNA splicing site. Said expression vector can beincorporated in COS cells by the DEAE-dextran method (Luthman, H. andMagnusson, G. (1983) Nucleic Acids Res. 11, 1295-1308), the calciumphosphate-DNA coprecipitation method (Graham, F. L. and van der Ed, A.J. (1973) Virology 52, 456-457), electroporation (Neumann E. et al.,(1982) EMBO J., 1, 841-845) and so forth, thus enabling a cell to beobtained that has been transfected by the desired vector.

In addition, in the case of using CHO cells as the host cell, byco-tranfecting with an expression vector and a vector able to express aneo-gene that functions as a G418-resistant marker, examples of whichinclude pRSVneo (Sambrook, J. et al. (1989): "Molecular Cloning--ALaboratory Manual", Cold Spring Harbor Laboratory, New York), pSV2-neo(Southern, P. J. and Berg, P. (1982) J. Mol. Appl. Genet. 1, 327-341)and so forth, and then selecting G418-resistant colonies, transfectedcells can be obtained that stably produce the ribozymepolyribonucleotide of the present invention.

Cells transfected with the desired vector obtained as described abovecan be cultured in accordance with conventional methods, and ribozymepolyribonucleotide can be produced within the cells by said culturing.Various types of media routinely used corresponding to the host cellsbeing employed can be suitably selected for the medium used in saidculturing. Examples of media that can be used in the case of theabove-mentioned COS cells include that to which serum components such asfetal bovine serum (FBS) have been added as necessary to media such asRPMI-1640 medium and Dulbecco's modified Eagle's minimum essentialmedium (DMEM).

The present invention also relates to a process for specificallycleaving a substrate polyribonucleotide using a ribozymepolyribonucleotide. Polyribonucleotide that has a detrimental effect onthe living body can be specifically cleaved by carrying out the processof the present invention in the living body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an analytical diagram of the substrate polyribonucleotidecleavage reaction by homochromatography.

FIG. 2 is a diagram showing the construction of the plasmid pRZ4Δneo.

FIG. 3 is a nucleotide sequence chart of a double-strand ribozymepolyribonucleotide gene (upper sequence is SEQ ID NO: 18 and lowersequence is SEQ ID NO: 19).

FIG. 4 is a diagram showing the construction of plasmid pRSV-rv12neo.

FIG. 5 is an analytical diagram of northern hybridization of NIH3T3cells co-transfected with plasmids pRZ4Δneo and pRSV-rv12neo.

FIG. 6 is an analytical diagram of a substrate polyribonucleotide byhomochromatography.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described below in more detail by way ofexamples and reference examples, but the present invention is notlimited to these.

EXAMPLE 1

Synthesis of Polyribonucleotide

The following polyribonucleotides (b), (c), (e) and (f) were synthesizedby a DNA automatic synthesizer (ABI, Model 394 DNA/RNA Synthesizer)using nucleoside 3'-phosphoroamidites wherein the 5'-hydroxyl group isprotected with a dimethoxytrityl group and the 2'-hydroxyl group isprotected with a tert-butyldimethylsilyl group (purchased from AmericanBionetics Inc.). ##STR9##

The RNA fragment was synthesized on a 1 μmol scale.

After completion of the synthesis, a CPG (controlled pore glass) towhich the synthesized oligonucleotide was coupled, was treated at roomtemperature for 1 hour with a mixed solution of concentrated ammoniawater and ethanol (3:1 v/v). After distilling off the solvent, 5 ml ofethanolic saturated ammonia was added, followed by warming at 55° C. for16 hours. The solvent was distilled off and 1 ml of a solution of 1MTBAF (tetrabutylammonium fluoride) in THF (tetrahydrofuran) was added tothe remaining solution, followed by stirring at room temperature for 24hours. After 5 ml of 0.1M triethylammonium acetate (pH 7.0) was added tothe mixture, C18 silica gel (Waters) column chromatography was performed(column size: 0.7×15 cm; eluted according to concentration gradientusing a solvent of 5 to 40% CH₃ CN and 50 mM triethylammoniumbicarbonate). Fractions having the coloring of dimethoxytrityl thateluted with CH₃ CN at a concentration of about 30% were collected,following by the addition of 5 ml of 0.01N HCl and stirring for 1 hour.After the mixture was neutralized with 0.1N aqueous ammonia, the aqueouslayer was washed with ethyl acetate. After distilling off the solvent,the residue was dissolved in 1.2 ml of sterile water. Thepolyribonucleotides contained in this fraction were separated andpurified with reverse phase HPLC and ion exchange HPLC.

Reverse phase HPLC was performed using an Inertsil ODS-2 column (φ10×250mm, GL Sciences, Inc.) by the linear concentration gradient method using0.1M triethylammonium acetate containing 5% CH₃ CN (pH 7.0) for solutionA and 0.1M triethylammonium acetate containing 25% CH₃ CN (pH 7.0) forsolution B.

In addition, ion exchange HPLC was performed using a TSK gel DEAE 2SWcolumn (φ4.6×250 mm, Tosoh Co., Ltd.) by the linear concentrationgradient method using 20% CH₃ CN/H₂ O for solution A and 2M HCOONH₄solution containing 20% CH₃ CN for solution B. The percentages ofsolution B used for reverse phase HPLC and ion exchange HPLC elution andretention times are shown in Table 1 below for each polyribonucleotide.

                  TABLE 1                                                         ______________________________________                                                     Total time of linear                                                                         Retention                                         B%           concentration gradient                                                                       time                                              ______________________________________                                        Reverse Phase HPLC                                                            (b) 15% -→ 35%                                                                          (20 minutes)   24.6 minutes                                  (c) 10% -→ 50%                                                                          (20 minutes)   21.4 minutes                                  (e) 25% -→ 50%                                                                          (20 minutes)   15.7 minutes                                  (f) 25% -→ 45%                                                                          (20 minutes)   16.5 minutes                                  Ion Exchange HPLC                                                             (b) 30% -→ 50%                                                                          (20 minutes)   12.4 minutes                                  (c) 30% -→ 50%                                                                          (20 minutes)   12.7 minutes                                  (e) 30% -→ 50%                                                                          (20 minutes)   12.9 minutes                                  (f) 30% -→ 50%                                                                          (20 minutes)   13.4 minutes                                  ______________________________________                                    

Reference Example 1

Synthesis of Polyribonucleotide

The following polyribonucleotides (a) and (d) (SEQ ID NO: 3) weresynthesized, separated and purified according to the method described inExample 1. ##STR10##

These polyribonucleotides were used in the control experiment describedlater as polyribonucleotides having low ribozyme activity.

In addition, the following polyribonucleotides (g) and (h)(SEQ ID NO: 6)were synthesized, separated and purified according to the methoddescribed in Example 1. ##STR11##

These polyribonucleotides were used in an experiment described later assubstrates of a ribozyme cleavage reaction.

The percentages of solution B used for reverse phase HPLC and ionexchange HPLC elution and retention times are shown in Table 2 for eachpolyribonucleotide.

                  TABLE 2                                                         ______________________________________                                                      Total time of linear                                                                         Retention                                        B%            concentration gradient                                                                       time                                             ______________________________________                                        Reverse Phase HPLC                                                            (a) (Reverse phase HPLC was not performed)                                                                 --                                               (g) 10% -→ 40%                                                                           (20 minutes)   19.0 minutes                                 (d) 25% -→ 50%                                                                           (20 minutes)   14.0 minutes                                 (h) 10% -→ 35%                                                                           (20 minutes)   18.7 minutes                                 Ion Exchange HPLC                                                             (a) 20% -→ 50%                                                                           (30 minutes)   22.5 minutes                                 (g) 20% -→ 40%                                                                           (20 minutes)   17.8 minutes                                 (d) 30% -→ 50%                                                                           (20 minutes)   15.0 minutes                                 (f) 20% -→ 40%                                                                           (20 minutes)   16.3 minutes                                 ______________________________________                                    

EXAMPLE 2

Labeling of 5' Terminal of Substrate RNA

[γ-³² P]ATP (0.5 μl, 5 μci), a buffer (1 μl, 250 mM Tris-HCl (pH 7.6),50 mM magnesium chloride, 50 mM mercaptoethanol), T4 polynucleotidekinase (0.5 μl, 1 unit, Takara Shuzo Co., Ltd.) and sterile water (3 μl)were added to either polyribonucleotide (g) or (h) described above (100pmol) followed by incubating for 1 hour at 37° C. Unreacted [γ-³² P]ATPand salt were removed using NENSORB20 (Dupont Corporation). The residuewas then dissolved in sterile water to obtain the 5'-labelledoligonucleotide.

EXAMPLE 3

Cleavage of Substrate Polynucleotide

The cleavage reaction of polyribonucleotide (a), polyribonucleotide (b)or polyribonucleotide (c) on polyribonucleotide (g) was carried out inthe following manner.

Polyribonucleotide (g) labelled at its 5'-terminal was dissolved in abuffer (25 mM MgCl₂, 40 mM Tris-HCl (pH 7.5) and 20 mM NaCl). Theribozyme oligonucleotide was added to the solution to start the reactionso that the final concentration of substrate was 1 μM and the finalconcentration of ribozyme was 0.5 μM. After the mixture was warmed at37° C. and a predetermined period of time elapsed, the reaction wasstopped by mixing with EDTA, namely by sampling a portion of thereaction mixture so as to make the final concentration of EDTA 50 mM.The changes in substrate decomposition were then observed with passageof time. The cleavage products were separated by homochromatography andthe cleavage rate was determined using a bioimage analyzer (Fuji PhotoFilm Co., Ltd.). The respective reaction forms are shown below.##STR12##

The results are shown in FIG. 1.

The polyribonucleotide having the oligonucleotide sequence of (g) hasalready been reported to be cleaved by (a) (Nucleic Acids Res. 17,7059-7071 (1989)). Lane 1 in FIG. 1 represents (g) before the reaction.Lanes 2 through 5 represent the products of cleavage of (g) by (a),while lanes 6 through 9 represent the products of cleavage of (g) by(b). The cleavage products are respectively shown for 15, 30, 60 and 90minutes after the start of the reaction. Due to the short chain lengthof the cleaved fragments, they are developed more quickly than theuncleaved (g) in homochromatography. In FIG. 1, these are shown at alocation above the location of the uncleaved (g). In addition, (a) and(b) catalytically cleaved (g). The values for Km (Michaelis constant)and Kcat (reaction rate constant), which are used as parameters forthis, were calculated using the Hanes-Woolf Plot. Those results areshown in Table 3.

                  TABLE 3                                                         ______________________________________                                                                        Kcat/Km                                       Ribozyme                                                                              Km (μM)   Kcat (min.sup.-1)                                                                        (μM.sup.-1 min.sup.-1)                     ______________________________________                                        (a)     1.1          0.12       1.1 × 10.sup.-1                                                         (100)*                                        (b)     0.63         0.08       1.3 × 10.sup.-1                                                         (118)*                                        ______________________________________                                         *Relative value when the ribozyme catalytic activity of (a) is taken to b     100.                                                                     

As is shown in Table 3, the ribozyme wherein a thermodynamically stable5'CUUCGG3' loop is introduced into the ribozyme polyribonucleotidesequence (b) demonstrates increased cleavage activity in comparison withthat of (a).

In addition, the cleavage reaction of polyribonucleotide (d),polyribonucleotide (e) or polyribonucleotide (f) on polyribonucleotide(h) was also carried out in a buffer (25 mM MgCl₂, 40 mM Tris-HCl (pH7.5) and 20 mM NaCl) so that the final concentration of substrate was 1μM and the final concentration of ribozyme polyribonucleotide was 0.4μM. The time in which half of the substrate was cleaved was thencompared. The respective reaction forms were as shown below. ##STR13##

Those results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Ribozyme      t.sub.1/2 (min)                                                                        Specific Activity                                      ______________________________________                                        (d)           21       1                                                      (e)            7       3                                                      (f)           15       1.4                                                    ______________________________________                                    

Both (e) and (f) have thermodynamically stable loops in the form of5'CUUCGG3', and their cleavage activities were higher than that of (d)which does not have a stable loop.

EXAMPLE 4

Construction of a Plasmid Having a Variant c-Ha-ras Gene wherein GGU ofCodon 12 of Normal c-Ha-ras Gene is Changed to GUU (pRSV-rv12neo), and aPlasmid Having a Double-Strand DNA that Codes for a RibozymePolyribonucleotide that Cleaves Variant ras mRNA (pRZ4Δneo)

Point mutation of either of codons 12, 13 or 61 occurs in c-Ha-ras genedetected from human cancer (Nature (1982) 300, 143-149). When thisvariant c-Ha-ras gene is transfected into NIH3T3 cells, it is known thatthe variant c-Ha-ras gene is expressed and the cells are transformed(Nature (1982) 300, 143-149). It is thought that by cleaving the mRNA ofan ras gene, wherein GGU of codon 12 of a normal c-Ha-ras gene ischanged to GUU (Jpn. J. Cancer Res. (1989) 80, 200-203), using aribozyme polyribonucleotide, expression of the variant ras gene in theNIH3T3 cells can be suppressed. The following provides a description ofthe construction of plasmid pRSV-rv12neo, having an ras gene wherein GGUof codon 12 of a normal c-Ha-ras gene is changed to GUU, and plasmidpRZ4Δneo, having a gene that codes for a ribozyme polyribonucleotidethat cleaves that ras mRNA.

FIG. 2 indicates a summary of construction of pRZ4Δneo. 3 μg ofpRSV-rg12 (Jpn. J. Cancer Res. (1989) 80, 200-20) was digested bywarming overnight at 37° C. with ClaI (15 U, Takara Shuzo Co., Ltd.) andSalI (30 U, Takara Shuzo Co., Ltd.). After ethanol precipitation, thedigestion product was dissolved in 0.1M Tris-HCl (pH 8.0, 48 μl),followed by addition of alkaline phosphatase derived from E. Coli (0.7U, Takara Shuzo Co., Ltd.) and warming at 37° C. for 2 hours. After themixture was treated with phenol-chloroform, a digestion product wasobtained by ethanol precipitation. A ribozyme polyribonucleotide genefragment formed into two strands by annealing (the base sequence isshown in FIG. 3, 0.2 pmol, 1 μl), a reaction buffer (4 μl, 330 mMTris-HCl (pH 7.6), 33 mM MgCl₂), 0.2M 2-mercaptoethanol (1 μl) and T4DNA ligase (1 μl, 350 U, Takara Shuzo Co., Ltd.) were added to the ClaIand SalI digestion products of pRSV-rg12 (1.5 μg, 0.5 pmol, 8 μl),followed by warming at 20° C. for 1.5 hours and precipitation withethanol to obtain pRZ4Δneo.

The resulting pRZ4ΔAneo was transfected into E. coli HB101 straintreated with Ca²⁺ and cultured overnight in agar media containingampicillin. Screening was the performed by rapid boiling method (Anal.Biochem. (1981) 114, 193-197) to isolate E. coli having pRZ4Δneo. ThisE. coli was then cultured and the pRZ4Δneo was isolated and purifiedaccording to the method of Norgard (Anal. Biochem. (1981) 113, 34-42).

FIG. 4 indicates a summary of the construction of pRSV-rv12neo. pMAMneo(3 μg, Clontech Corporation) was digested by warming overnight at 37° C.with ClaI (10 U, Takara Shuzo Co., Ltd.) and SalI (20 U, Takara ShuzoCo., Ltd.). The mixture was dissolved in 0.1M Tris-HCl (pH 8.0, 48 μl)and to the resulting solution was added alkaline phosphatase derivedfrom E. Coli (0.7U, Takara Shuzo Co., Ltd.), followed by warming at 37°C. for 2 hours. After the mixture was treated with phenol-chloroform, adigestion product was obtained by ethanol precipitation. pRSV-rv12 (Jpn.J. Cancer Res. (1989) 80, 200-203) was similarly digested with ClaI andSalI, and then the variant c-Ha-ras gene fragment was separated bylow-melting point agarose gel electrophoresis. The pMAMneo ClaI and SalIdigestion products along with the variant c-Ha-ras gene fragment wereligated with T4 DNA ligase to obtain pRSV-rv12neo. This was thenisolated and purified by a method similar to that used for isolatingpRZ4Δneo.

EXAMPLE 5

Introduction of pRSV-rv12neo and pRZ4Δneo into NIH3T3 Cells byCo-Transfection and Isolation of Cells Exhibiting Morphology Identicalto Normal Cells

NIH3T3 cells transfected with pRSV-rv12neo having a variant c-Ha-rasgene, in which GGU of codon 12 of a normal c-Ha-ras gene is changed toGUU, are known to be transformed. It is thought that the transcriptionproduct of the ras gene can be cleaved to return the transformed cellsto a form identical to normal cells by expressing a gene that codes forribozyme polyribonucleotide. The process will be described below.

pRSV-rv12neo (10 ng), pRZ4Δneo (2 μg) and NIH3T3 cell DNA as carrier DNA(30 μg) were transfected into NIH3T3 cells (8×10⁵ cells) by the calciumphosphate method (Virology (1973) 52, 456-457). Two days later, thecells were cultured for about 1 week in DMEM media (containing 5% bovineserum) to which Geneticin (300 mg/500 ml, Gilbco Corporation) had beenadded. Those colonies that exhibited morphology identical to that ofnormal cells were detected microscopically. Those colonies were isolatedusing penicillin caps.

The introduction of pRSV-rv12neo into NIH3T3 cells has been confirmed bythe PCR method (Science (1989) 239, 487-491). The following two primerswere synthesized with the model 394 DNA/RNA automatic synthesizer of theABI Corporation to be used. Namely, these consisted of an upper primer(CGATATGACCGAATACAAACT) (SEQ ID NO: 20) and a lower primer(TCGAGTATCAGCCTGGGCCAGATTCGTCCGGCGGGTTAGCT)(SEQ ID NO: 21).Incidentally, the literature of Miura et al. (Jpn. J. Cancer Res. (1986)77, 45-51) was referred to for the nucleotide sequences of the primersused here.

EXAMPLE 6

Confirmation of Ribozyme Effect by Northern Hybridization

After culturing the cells exhibiting identical morphology to normalcells isolated using the process of Example 5 in DMEM medium, the cellswere collected by centrifugation and treated with RNAzol (Cinna BiotechCorporation) containing guanidinium thiocyanate to extract total RNA.

The total RNA obtained from the cells (approximately 20 μg) was appliedto 1% agarose gel electrophoresis containing formaldehyde. This wastransferred to a nitrocellulose filter and baked at 80° C. for 2 hoursunder reduced pressure to fix the RNA. The filter was immersed in 20 mlof pre-hybridization solution (5× SSC, 2× Denhardt, 1% SDS, 100 mg/mlsalmon sperm DNA, 50 mM sodium phosphate (pH 6.7), 50% formamide) andshaken at 42° C. for 8 hours, c-Ha-ras gene, in which codon 12 hadchanged to GUU, was labeled by a random primed DNA labeling kit(Boehringer-Mannheim GmbH.), and this labeled form was added as a probe(1.1×10⁹ dpm/μg) to 10 ml of the above solution followed by shaking at42° C. for 17 hours. After washing the filter with a solution of 0.2×SSC-0.5% SDS at 60° C., it was then analyzed with a bioimage analyzer(Fuji Photo Film Co., Ltd.). Human β-actin mRNA was used for theinternal standard.

NIH3T3 cells transformed only with pRSV-rv12neo were selected for use asthe positive control cells that express the variant c-Ha-ras gene. Inaddition, normal NIH3T3 cells were selected for use as the negativecontrol cells that do not retain the variant c-Ha-ras gene. Total RNAwas extracted from both types of cells using a similar process as thatdescribed above to attempt hybridization.

FIG. 5 indicates the results of northern hybridization. Lane 1 is themRNA derived from NIH3T3 cells transformed with variant ras gene(pRSV-rv12neo). Lane 2 is the mRNA derived from normal NIH3T3 cells.Lanes 3 and 4 show the results of analysis of mRNA derived from coloniesof cells co-transfected with pRZ4Δneo and pRSV-rv12neo that exhibitedmorphology identical to normal cells. Since ras mRNA was not able to bedetected in lanes 3 and 4, ribozyme polyribonucleotide was thought tohave cleaved that ras mRNA within the cells. This finding indicates thatribozyme polyribonucleotide suppresses the transformation of cellscaused by expression of a variant ras gene.

EXAMPLE 7

Synthesis of Polyribonucleotide that Cleaves SubstratePolyribonucleotide Having a Base Sequence of Human ImmunodeficiencyVirus (HIV) RNA

The following polyribonucleotides (i) and (j), which cleave a substratepolyribonucleotide having an HIV RNA base sequence, were synthesized bya DNA automatic synthesizer (Cyclon Plus DNA/RNA Synthesizer, JapanMillipore Limited) using nucleoside 3'-phosphoramidites (Japan MilliporeLimited) wherein the 5'-hydroxyl group is protected with adimethoxytrityl group and the 2'-hydroxyl group is protected with atert-butyldimethylsilyl group. ##STR14##

The RNA fragment was synthesized on a 1 μmol scale.

After completion of synthesis, a CPG (controlled pore glass), to whichthe synthesized oligonucleotide was coupled, was treated at roomtemperature for 2 hours with a mixed solution of concentrated ammoniawater and ethanol (3:1 v/v), followed by warming at 55° C. for 16 hours.The solvent was distilled off and 1 ml of a 1M solution of TBAF(tetrabutylammonium fluoride) in THF (tetrahydrofuran) was added to theresidue followed by stirring at room temperature for 24 hours. After 5ml of 0.1M triethylammonium acetate (pH 7.0) was added to the mixture,C18 silica gel (Waters) column chromatography was performed (columnsize: 1.5×12 cm; eluted according to concentration gradient using asolvent of 20-40% CH₃ CN and 50 mM triethylammonium bicarbonate).Fractions having the coloring of dimethoxytrityl that eluted with CH₃ CNat a concentration of about 30% were collected, followed by the additionof 5 ml of 0.01N HCl and stirring for 1 hour. After neutralizing with0.1N aqueous ammonia, the aqueous layer was washed with ethyl acetate.After distilling off the solvent, the product was dissolved in 3 ml ofsterile water. The polyribonucleotides contained in this fraction wereseparated and purified with ion exchange HPLC.

Ion exchange HPLC was performed using a TSK gel DEAE 2SW colum (φ4.6×250mm, Tosoh Co., Ltd.) by the linear concentration gradient method using20% CH₃ CN/H₂ O for solution A and 2M HCOONH₄ aqueous containing 20% CH₃CN for solution B. The percentages of solution B used for the ionexchange HPLC elution and retention times are shown in Table 5 below foreach polyribonucleotide.

                  TABLE 5                                                         ______________________________________                                                      Total Time of                                                                 Linear                                                                        Concentration                                                   B %           Gradient    Retention                                           ______________________________________                                        (i): 20% -→ 60%                                                                      20 minutes  18.6 minutes                                        (j): 20% -→ 60%                                                                      20 minutes  17.9 minutes                                        ______________________________________                                    

Reference Example 2

Synthesis of Polyribonucleotide Having an HIV RNA Base Sequence

Polyribonucleotide (k), having an HIV RNA base sequence (19 mer havingsequences from nucleotide no. 106 to no. 124 described in theliterature: Nature 313, 450-458 (1985)), polyribonucleotide (1) (10 merhaving sequences from nucleotide no. 115 to no. 124 described in theabove-mentioned literature) and polyribonucleotide (m) (6 mer havingsequences from nucleotide no. 119 to no. 124 described in theabove-mentioned literature) were synthesized, separated and purifiedaccording to the method described in Example 7. ##STR15##

Polyribonucleotide (k) was used in an experiment described hereinafteras the substrate for a ribozyme cleavage reaction. In addition,polyribonucleotides (1) and (m) were used in an experiment describedhereinafter as controls for compounds produced in a ribozyme cleavagereaction. The percentages of solution B used for ion exchange HPLCelution and retention times are shown in Table 6 for eachpolyribonucleotide.

                  TABLE 6                                                         ______________________________________                                                      Total Time of                                                                 Linear                                                                        Concentration                                                   B %           Gradient    Retention                                           ______________________________________                                        (k): 10% -→ 60%                                                                      20 minutes  19.1 minutes                                        (l): 10% -→ 60%                                                                      20 minutes  17.1 minutes                                        (m): 10% -→ 60%                                                                      20 minutes  15.1 minutes                                        ______________________________________                                    

EXAMPLE 8

Cleavage of Polyribonucleotide Having an HIV RNA Base Sequence by aPolyribonucleotide Having Ribozyme Activity

The cleavage reactions of polyribonucleotide (i) on polyribonucleotide(k), and of polyribonucleotide (j) on polyribonucleotide (k) werecarried out as described below.

Substrate polyribonucleotide (k) was dissolved in a buffer (25 mM MgCl₂,40 mM Tris-HCl (pH 7.5) and 20 mM NaCl). The ribozyme polyribonucleotidewas added to start the reaction so that the final concentration ofsubstrate was 1.25 μM and the final concentration of ribozyme was 0.063μM. After the mixture was warmed at 37° C. for 1 hour, the cleavagereaction was analyzed by ion exchange HPLC (TSK gel DEAE 2SW, 4.6×250mm, 20% CH₃ CN/H₂ O for solution A, 2M aqueous HCOONH₄ containing 20%CH₃ CN for solution B: linear concentration gradient of 10-→60%/20minutes, flow rate: 1 ml/min). The respective reaction forms are shownbelow. ##STR16##

In the case of reacting polyribonucleotide (i) on polyribonucleotide (k)under the above-mentioned conditions, a decrease was observed in thepeak of polyribonucleotide (k) in ion exchange HPLC. In contrast, twopeaks appeared at 17.1 minutes and 17.3 minutes. Since the peak having aretention time of 17.1 minutes coincided with the retention time ofpolyribonucleotide (1), it was confirmed that cleavage took place at thetarget location. In addition, determination of the cleavage rate fromthe area value obtained from HPLC of the peak of polyribonucleotide (k)after the cleavage reaction as well as the two peaks that appearedyielded a value of 90%.

In the case of reacting polyribonucleotide (j) on polyribonucleotide (k)under the above-mentioned conditions, a decrease was observed in thepeak of polyribonucleotide (k) in ion exchange HPLC. In contrast, twopeaks appeared at 15.1 minutes and 18.2 minutes. Since the peak having aretention time of 15.1 minutes coincided with the retention time ofpolyribonucleotide (m), it was confirmed that cleavage took place at thetarget location. In addition, determination of the cleavage rate fromthe area value obtained from HPLC of the peak of polyribonucleotide (k)after the cleavage reaction as well as the two peaks that appearedyielded a value of 88%.

EXAMPLE 9

Synthesis of Polyribonucleotide

The following polyribonucleotide (n) was synthesized by a DNA automaticsynthesizer (Model 394 DNA/RNA Synthesizer, Applied Biosystems, Inc.)using nucleoside 3'-phosphoramidites wherein the 5'-hydroxyl group isprotected with a dimethoxytrityls group and the 2'-hydroxyl group isprotected with a tert-butyldimethylsilyl group (purchased from AmericanBiotics, Inc.). ##STR17##

The RNA fragment was synthesized on a 1 μmol scale.

After completion of synthesis, a CPG (controlled pore glass), to whichthe synthesized oligonucleotide was coupled, was treated at roomtemperature for 1 hour with a mixed solution of concentrated ammoniawater and ethanol (3:1 v/v). After the solvent was distilled off, 5 mlof ethanolic saturated ammonia was added to the residue, followed bywarming at 55° C. for 16 hours. The solvent was distilled off and 1 mlof a 1M solution of TBAF (tetrabutylammonium fluoride) in THF(tetrahydrofuran) was added to the remaining solution followed bystirring at room temperature for 24 hours. After 5 ml of 0.1Mtriethylammonium acetate (pH 7.0) was then added to the mixture, C18silica gel (Waters) column chromatography was performed (column size:0.7×15 cm; eluted according to concentration gradient using a solvent of5 to 40% CH₃ CN and 50 mM triethylammonium bicarbonate). Fractionshaving the coloring of dimethoxytrityl that eluted with CH₃ CN at aconcentration of about 30% were collected, followed by the addition of 5ml of 0.01N HCl and stirring for 1 hour. After the mixture wasneutralized with 0.1N aqueous ammonia, the aqueous layer was washed withethyl acetate. After the solvent was distilled off, the residue wasdissolved in 1.2 ml of sterile water. The polyribonucleotides containedin this fraction were separated and purified with reverse phase HPLC andion exchange HPLC.

The reverse phase HPLC was performed using an Inertsil ODS-2 column(φ10×250 mm, GL Sciences Inc.) by the linear concentration gradientmethod using 0.1M triethylammonium acetate containing 5% CH₃ CN (pH 7.0)for solution A and 0.1M triethylammonium acetate containing 25% CH₃ CN(pH 7.0) for solution B.

In addition, the ion exchange HPLC was performed using a TSK gel DEAE2SW column (φ4.6×250 mm, Tosoh Co., Ltd.) by the linear concentrationgradient method using 20% CH₃ CN/H₂ O for solution A and 2M HCOONH₄containing 20% CH₃ CN for solution B. The percentages of solution B usedfor the reverse phase HPLC and the ion exchange HPLC elution andretention times are shown in Table 7 below for polyribonucleotide (n).

                  TABLE 7                                                         ______________________________________                                                             Total Time of                                                                 Linear                                                   Type of              Concentration                                            Analysis  B %        Gradient    Retention Time                               ______________________________________                                        Reverse phase                                                                           10% -→ 50%                                                                        20 minutes  18.1 minutes                                 HPLC                                                                          Ion exchange                                                                            30% -→ 50%                                                                        20 minutes  20.0 minutes                                 HPLC                                                                          ______________________________________                                    

In addition, the following polyribonucleotides (o) and (p) have alreadybeen reported by Sekiguchi et al. (Nucleic Acids Res. 19, 6833-6838(1991)). ##STR18##

EXAMPLE 10

Labeling of 5' Terminal of Substrate Polyribonucleotide (P)

[γ-³² P]ATP (0.5 μl, 5 μci), a buffer (1 μl, 250 mM Tris-HCl (pH 7.6),50 mM magnesium chloride, 50 mM 2-mercaptoethanol), T4polyribonucleotide kinase (0.5 μl, 1 unit, Takara Shuzo Co., Ltd.) andsterile water (3 μl) were added to the above-mentionedpolyribonucleotide (p) (100 pmol) followed by incubating at 37° C. for 1hour. Unreacted [γ-³² P]ATP and salt were removed using NENSORB20(Dupont Corporation). The residue was then dissolved in sterile water toobtain the 5'-labelled oligonucleotide.

EXAMPLE 11

Cleavage of Substrate Polyribonucleotide

The cleavage reaction of polyribonucleotide (n) or (o) on substratepolyribonucleotide (p) was carried out in the following manner.

Polyribonucleotide (p) (1.62 pmol) labelled at its 5'-terminal wasdissolved in 10 μl of a buffer (40 mM Tris-HCl (pH 7.5), 12 mM MgCl₂ and2 mM spermidine 3 HCl). In addition, after ribozyme polyribonucleotide(n) or (o) (0.64 pmol) was dissolved in 10 μl of the same buffer, eachsolution was warmed at 65° C. for 2 minutes, followed by cooling withwater. The ribozyme solutions were then added to the substrate to startthe reaction.

After a predetermined period of time had elapsed, the reaction wasstopped by sampling 2 μl of the reaction solution in a solutioncontaining 2 μl of 50 mM EDTA. The changes in substrate decompositionwere then observed with passage of time. The cleavage products wereseparated by homochromatography and the cleavage rate was determinedusing a bioimage analyzer (BAS2000 System, Fuji Photo Film Co., Ltd.).The respective reaction forms are shown below. ##STR19##

The results in the case of a reaction temperature of 42° C. are shown inFIG. 6.

It has already been reported that polyribonucleotide (p) is cleaved bypolyribonucleotide (o) having a naturally-occurring base sequence(Nucleic Acids Res. 19, 6833-6838 (1991)).

In FIG. 6, lane X6 indicates (p) before reaction. Lanes 1 through 5 arethe products of cleavage of (p) by (o), while lanes 7 through 11 are theproducts of cleavage of (p) by (n). The cleavage products arerespectively shown for 3, 9, 12, 15 and 18 minutes after the start ofthe reaction.

Due to the short chain length of the cleaved fragments, they aredeveloped more quickly than the uncleaved (p) in homochromatography. InFIG. 6, the cleaved fragments are shown at a higher position than theuncleaved (p).

In addition, the time in which half of substrate (P) is cleaved byeither (n) or (o) (t1/2) was determined. Those results are shown inTable 8 together with those for cleavage reaction at 32°, 37° and 47° C.

                  TABLE 8                                                         ______________________________________                                                       t1/2when using                                                                            t1/2when using                                     Temperature (°C.)                                                                     (n) (minutes)                                                                             (o) (minutes)                                      ______________________________________                                        32             15          24                                                 37             4            8                                                 42             2           10                                                 47             8           54                                                 ______________________________________                                    

As shown in Table 8, a ribozyme polyribonucleotide (n), in which a5'CUUCGG3' thermodynamically stable loop is introduced into itssequence, demonstrated increased cleavage activity at temperatures from32° C. to 47° C. in comparison with the naturally-occuring form (o).

EXAMPLE 12

Synthesis of Polyribonucleotide (q) that Cleaves SubstratePolyribonucleotide Having the Base Sequence of Human ImmunodeficiencyVirus (HIV) RNA

The following polyribonucleotide (q), which cleaves a substratepolyribonucleotide having an HIV RNA base sequence, was synthesized by aDNA automatic synthesizer (Cyclone Plus DNA/RNA Synthesizer, JapanMillipore Limited) using nucleoside 3'-phosphoramidites (Japan MilliporeLimited) wherein the 5'-hydroxyl group is protected with adimethoxytrityl group and the 2'-hydroxyl group is protected with atert-butyldimethylsilyl group. ##STR20##

The RNA fragment was synthesized on a 1 μmol scale.

After completion of synthesis, a CPG (controlled pore glass), to whichthe synthesized oligonucleotide was coupled, was treated at roomtemperature for 2 hours with a mixture of concentrated ammonia water andethanol (3:1 v/v), followed by warming at 55° C. for 16 hours. Thesolvent was distilled off and 1 ml of a 1M solution of TBAF(tetrabutylammonium fluoride) in THF (tetrahydrofuran) was added to theresidue followed by stirring at room temperature for 24 hours. Afterthen adding 5 ml of 0.1M triethylammonium acetate (pH 7.0), C18 silicagel (Waters) column chromatography was performed (column size: 1.5×12cm; eluted according to concentration gradient using 20 to 40% CH₃ CNand an aqueous 50 mM triethylammonium bicarbonate as a solvent).Fractions having the coloring of dimethoxytrityl that eluted with CH₃ CNat a concentration of about 30% were collected, followed by the additionof 5 ml of 0.01 N HCl and stirring for 1 hour. After the mixture wasneutralized with 0.1N aqueous ammonia, the aqueous layer was washed withethyl acetate. After distilling off the solvent, the product wasdissolved in 3 ml of sterile water. The polyribonucleotides contained inthis fraction were separated with ion exchange HPLC, and thenadditionally separated and purified with reverse phase HPLC.

The ion exchange HPLC was performed using a TSK gel DEAE 2SW column(4.6×250 mm, Tosoh Co., Ltd.) by the linear concentration gradientmethod using 20% CH₃ CN/H₂ O for solution A and an aqueous 2M HCOONH₄containing 20% CH₃ CN for solution B (B 20%-→60% (20 minutes)).Polyribonucleotide (q) eluted at 20.6 minutes.

The reverse phase HPLC was performed using an Inertsil ODS column(6.0×150 mm, GL Sciences Inc.) by the linear concentration gradientmethod using 0.1M triethylammonium acetate (TEAA, pH 7.0) containing 5%CH₃ CN for solution A and 0.1M TEAA containing 25% CH₃ CN (pH 7.0) forsolution B (B 20%-→60% (20 minutes)). Polyribonucleotide (q) eluted at18.2 minutes.

Reference Example 3

Synthesis of Polyribonucleotide Having an HIV RNA Base Sequence

Polyribonucleotide (r), having an HIV RNA base sequence (19 mer havingsequences from nucleotide no. 106 to no. 124 described in theliterature: Nature 313, 450-458 (1985)), and polyribonucleotide (s) (13mer having sequences from nucleotide no. 112 to no. 124 described in theabove-mentioned literature) were synthesized, separated and purifiedaccording to the method described in Example 12.

    5'GUGOCCGUCUGUUGUGUGA3'                                    (r)(SEQ ID NO: 22)

    5'GUCUGUUGUGUGA3'                                          (s)(SEQ ID NO: 27)

Polyribonucleotide (r) was used in an experiment described hereinafteras the substrate for a hairpin ribozyme cleavage reaction. In addition,polyribonucleotide (s) was used in an experiment described hereinafteras a compound formed in a ribozyme cleavage reaction.

The percentages of solution B used for ion exchange HPLC elution andretention times, along with the percentages of solution B used forreverse phase HPLC elution and retention times are shown in Table 9 foreach polyribonucleotide (r) and (s).

                  TABLE 9                                                         ______________________________________                                                               Total Time of                                                                 Linear Con-                                            Type of                centration Retention                                   Analysis    B %        Gradient   Time                                        ______________________________________                                        (r) Reverse     10% -→ 50%                                                                        20 minutes                                                                             14.8 minutes                                  phase HPLC                                                                    Ion exchange                                                                              10% -→ 60%                                                                        20 minutes                                                                             19.1 minutes                                  HPLC                                                                      (s) Reverse     10% -→ 50%                                                                        20 minutes                                                                             12.2 minutes                                  phase HPLC                                                                Ion exchange                                                                              Ion exchange HPLC was not performed.                              HPLC                                                                          ______________________________________                                    

EXAMPLE 13

Cleavage of Polyribonucleotide Having an HIV RNA Base Sequence by aPolyribonucleotide Having Ribozyme Activity

The cleavage reaction of polyribonucleotide (q) on polyribonucleotide(r) was carried out as described below.

Substrate polyribonucleotide (r) (250 pmol) was dissolved in 100 μl of abuffer (40 mM Tris-HCl (pH 7.5), 12 mM MgCl₂ and 2 mM Spermidine.3HCl).In addition, the ribozyme polyribonucleotide (q) (12.5 pmol) wasdissolved in 100 μl of the same buffer. Both solutions were respectivelywarmed at 65° C. for 2 minutes followed by cooling with ice. Thereaction was then started by adding the ribozyme solution to thesubstrate. After the mixture was warmed at 37° C. for 1 hour, thereaction was stopped by addition of 50 μl of 50 mM EDTA. The cleavagereaction was analyzed by reverse phase HPLC (Inertsil ODS, 6.0×150 mm,linear concentration gradient method using 0.1M TEAA containing 5% CH₃CN (pH 7.0) for solution A and 0.1M TEAA containing 25% CH₃ CN (pH 7.0)for solution B, B 10%-→50%/20 minutes, flow rate: 1 ml/min). Therespective reaction forms are shown below. ##STR21##

In the case of reacting polyribonucleotide (q) on polyribonucleotide (r)under the above-mentioned conditions, a decrease was observed in thepeak of polyribonucleotide (r) in reverse phase HPLC. In contrast, apeak appeared at 12.2 minutes. Since this peak coincided with theretention time of polyribonucleotide (s), it was confirmed that cleavagetook place at the target location. In addition, determination of thecleavage rate from the area value obtained from HPLC of the peak ofpolyribonucleotide (r) after the cleavage reaction as well as the peakthat appeared at 12.2 minutes yielded a value of 97%.

Industrial Applicability

The ribozyme polyribonucleotide of the present invention can beadministered directly with a carrier that can be used pharmacologicallyin animals, plants and humans. Examples of its administration formsinclude oral administration in the form of tablets, capsules, granules,powders or syrups or parenteral administration in the form ofinjections, intravenous infusion or suppositories.

In addition, the ribozyme polyribonucleotide of the present inventioncan also be administered by enclosing in a transporter such as aliposome.

Although the dose varies according to symptoms, age, body weight and soforth, it is ordinary administered to an adult in an amount of about 0.1mg to 1000 mg per day in the case of oral administration, and this dosemay be given in a single administration or over the course of severalseparate administrations. In the case of parenteral administration, itis administered to an adult in an amount of 0.1 mg to 1000 mg perdosing, and this can be administered by subcutaneous injection,intramuscular injection or intravenous injection.

Further, the effect of the present invention can also be obtained byincorporating the DNA of the present invention into a suitable vectorand administering said vector into the body to express the ribozymepolyribonucleotide in cells. Retrovirus and vaccinia virus are examplesof such vectors.

Moreover, cells are isolated from the living body of an animal, plant orhuman, the said cells are transfected with the expression vector of thepresent invention, and the transfected cells are cultured to be giventhose cells the capacity to produce a desired ribozymepolyribonucleotide in those cells, followed by transplanting thesetransfected cells into the original donor body to impart resistance tothe specific polyribonucleotide to the living body. The use of thisprocess makes it possible to make the human body resistant to, forexample, polyribonucleotides associated with a specific disease such asAIDS, or polyribonucleotides related to a cancer gene.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 27                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CUACACCCUGAUGAGCCGCUUCGGCGGCGAAACAGCGC38                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CUACACCCUGAUGAGGCCGCUUCGGCGGCGAAACAGCGC39                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCCUAGCUGAUGAAGGGUGAUACCCUGAAACCA33                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCCUAGCUGAUGAGCCGCUUCGGCGGCGAAACCA34                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCCUAGCUGAUGAUGCCGCUUCGGCGGCGAAACCA35                                         (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GGUCCUAGGA10                                                                  (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CACAACACUGAUGAGCCGCUUCGGCGGCGAAACGGGCA38                                      (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       UCACACCUGAUGAGCCGCUUCGGCGGCGAAACAGAC36                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AAACAGAGAAGUCAACCAGAGAAACACACCUUCGGGUGGUAUAUUACCUGGUA53                       (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CACAACAAGAAGGCAACCAGAGAAACACACCUUCGGGUGGUAUAUUACCUGGUA54                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      NNNCUGANGANSSSCUUCGGSSSGAAANNN30                                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      NNNCUGANGASSSCUUCGGSSSGAAANNN29                                               (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      NNNUHNNN8                                                                     (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      NNNMGAANNNNACCAGAGAAACANNNCUUCGGNNNGUAUAUUACCUGGUA50                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      NNNNNGHHNNN11                                                                 (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CUACACCCUGAUGAGUCGUGAUACGACGAAACAGCGC37                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GCGCUGUUGGUGUAG15                                                             (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 43 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CGATCTACACCCTGATGAGCCGCTTCGGCGGCGAAACAGCGCG43                                 (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TCGACGCGCTGTTTCGCCGCCGAAGCGGCTCATCAGGGTGTAGAT45                               (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CGATATGACCGAATACAAACT21                                                       (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Other nucleic acid                                        (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TCGAGTATCAGCCTGGGCCAGATTCGTCCGGCGGGTTAGCT41                                   (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GUGCCCGUCUGUUGUGUGA19                                                         (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      UGUUGUGUGA10                                                                  (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 base pairs                                                      (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GUGUGA6                                                                       (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AAACAGAGAAGUCAACCAGAGAAACACACGUUGUGGUAUAUUACCUGGUA50                          (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      UGACAGUCCUGUUUC15                                                             (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: mRNA                                                      (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GUCUGUUGUGUGA13                                                               __________________________________________________________________________

We claim:
 1. A polyribonucleotide represented by the following formula(I): ##STR22## wherein, U represents an uracil nucleotide, C a cytosinenucleotide, A an adenine nucleotide, and G a guanine nucleotide,Srepresents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, V represents either an uracilnucleotide, adenine nucleotide, cytosine nucleotide or guaninenucleotide, Q₁ through Q_(n) may be the same or different from oneanother and represent either a cytosine nucleotide or guaninenucleotide, R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n), W₁ through W_(k) may be the same ordifferent from one another and represent either an uracil nucleotide,adenine nucleotide, cytosine nucleotide or guanine nucleotide, X₁through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, and k, m and n may be the same ordifferent from one another and represent an integer from 1 to
 10. 2. Thepolyribonucleotide in the formula (I) according to claim 1, wherein W₁is a cytosine nucleotide or a guanine nucleotide.
 3. Apolyribonucleotide represented by the following formula (II): ##STR23##wherein, U represents an uracil nucleotide, C a cytosine nucleotide, Aan adenine nucleotide, and G a guanine nucleotide,S represents either anuracil nucleotide, adenine nucleotide, cytosine nucleotide or guaninenucleotide, Q₁ through Q_(n) may be the same or different from oneanother and represent either a cytosine nucleotide or guaninenucleotide, R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n), W₁ through W_(k) may be the same ordifferent from one another and represent either an uracil nucleotide,adenine nucleotide, cytosine nucleotide or guanine nucleotide, X₁through X_(m) may be the same or different from one another andrepresent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, and k, m and n may be the same ordifferent from one another and represent an integer from 1 to
 10. 4. Thepolyribonucleotide of the formula (II) according to claim 3, wherein W₁is a cytosine nucleotide or a guanine nucleotide.
 5. A DNA that codesfor the polyribonucleotide according to claim 1, 2, 3 or
 4. 6. Arecombinant vector that includes the DNA according to claim
 5. 7. A hostcell transfected with the recombinant vector according to claim
 6. 8. Aprocess for cleaving a polyribonucleotide β(SEQ ID NO: 13) at a siteindicated with an arrow in the following formula (III) comprisingcontacting a substrate polyribonucleotide β in the formula (III) with aribozyme polyribonucleotide α (SEQ ID NO: 11) in the following formula(III): ##STR24## wherein, U represents an uracil nucleotide, C acytosine nucleotide, A an adenine nucleotide, and G a guaninenucleotide,S represents either an uracil nucleotide, adenine nucleotide,cytosine nucleotide or guanine nucleotide, V represents either an uracilnucleotide, adenine nucleotide, cytosine nucleotide or guaninenucleotide, Q₁ through Q_(n) are the same or different from one anotherand represent either a cytosine nucleotide or guanine nucleotide. R₁through R_(n) represent nucleotides that are respectively complementaryto Q₁ through Q_(n), W₁ through W_(k) are the same or different from oneanother and represent either an uracil nucleotide, adenine nucleotide,cytosine nucleotide or guanine nucleotide, X₁ through X_(m) are the sameor different from one another and represent either an uracil nucleotide,adenine nucleotide, cytosine nucleotide or quanine nucleotide, Prepresents either an uracil nucleotide, adenine nucleotide or cytosinenucleotide, Y₁ through Y_(k) represent nucleotides that are respectivelycomplementary to W₁ through W_(k), Z₁ through Z_(m) representnucleotides that are respectively complementary to X₁ through X_(m), andk, m and n are the same or different from one another and represent aninteger from 1 to
 10. 9. The process for cleaving the polyribonucleotideβ in formula (III) according to claim 8, wherein Y₁ is a guaninenucleotide or cytosine nucleotide complementary to W₁, wherein W₁ is acytosine nucleotide or guanine nucleotide.
 10. A process for cleaving apolyribonucleotide δ (SEQ ID NO: 13) at a site indicated with an arrowin the following formula (IV) comprising contacting a substratepolyribonucleotide δ in the following formula (IV) with a ribozymepolyribonucleotide γ (SEQ ID NO: 12) in the following formula (IV):##STR25## wherein, U represents an uracil nucleotide, C a cytosinenucleotide, A an adenine nucleotide, and G a guanine nucleotide,Srepresents either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, Q₁ through Q_(n) are the same ordifferent from one another and represent either a cytosine nucleotide orguanine nucleotide, R₁ through R_(n) represent nucleotides that arerespectively complementary to Q₁ through Q_(n), W₁ through W_(k) are thesame or different from one another and represent either an uracilnucleotide, adenine nucleotide, cytosine nucleotide or guaninenucleotide, X₁ through X_(m) are the same or different from one anotherand represent either an uracil nucleotide, adenine nucleotide, cytosinenucleotide or guanine nucleotide, P represents either an uracilnucleotide, adenine nucleotide or cytosine nucleotide. Y₁ through Y_(k)represent nucleotide that are respectively complementary to W₁ throughW_(k), Z₁ through Z_(m) represent nucleotides that are respectivelycomplementary to X₁ through X_(m), and k, m and n are the same ordifferent from one another and represent an integer from 1 to
 10. 11.The process for cleaving the polyribonucleotide δ (SEQ ID NO: 13) informula (IV) according to claim 10, wherein Y₁ isa guanine nucleotide orcytosine nucleotide complementary to W₁, wherein W₁ is a cytosinenucleotide or guanine nucleotide.
 12. A polyribonucleotide containingthe nucleotide sequence represented by the following formula (V) (SEQ IDNO: 14): ##STR26## wherein, U represents an uracil nucleotide, C acytosine nucleotide, A an adenine nucleotide, and G a guaninenucleotide,S represents either an adenine nucleotide or cytosinenucleotide, Q₁ through Q_(n) may be the same or different from oneanother and represent either an uracil nucleotide, adenine nucleotide,cytosine nucleotide or guanine nucleotide, R₁ through R_(n) representnucleotides that are respectively complementary to Q₁ through Q_(n), W₁through W₄ may be the same or different from one another and representeither an uracil nucleotide, adenine nucleotide, cytosine nucleotide orguanine nucleotide, X₁ through X_(m) may be the same or different fromone another and represent either an uracil nucleotide, adeninenucleotide, cytosine nucleotide or guanine nucleotide, and m and n maybe the same or different from each other and represent an integer from 1to
 10. 13. The polyribonucleotide in the formula (V) (SEQ ID NO: 14)according to claim 12, wherein S is an adenine nucleotide and n is 3.14. A process for cleaving a polyribonucleotide ζ (SEQ ID NO: 15), whichis included in the nucleotide sequence represented by a formula (VI), ata site indicated with an arrow in the formula (VI) comprising contactinga substrate polyribonucleotide in the following formula (VI) with aribozyme polyribonucleotide ε (SEQ ID NO: 14), which is included in thenucleotide sequence represented by the following formula (VI): ##STR27##wherein, U represents an uracil nucleotide, C a cytosine nucleotide, Aan adenine nucleotide, and G a quanine nucleotide,S represents either anadenine nucleotide or a cytosine nucleotide, Q₁ through Q_(n) are thesame or different from one another and represent either an uracilnucleotide, adenine nucleotide, cytosine nucleotide or guaninenucleotide, R₁ through R_(n) represent nucleotides that are respectivelycomplementary to Q₁ through Q_(n), W₁ through W₄ are the same ordifferent from one another and represent either an uracil nucleotide,adenine nucleotide, cytosine nucleotide or guanine nucleotide, X₁through X_(m) are the same or different from one another and representeither an uracil nucleotide, adenine nucleotide, cytosine nucleotide orguanine nucleotide, P represents either an uracil nucleotide, adeninenucleotide, cytosine nucleotide or guanine nucleotide, L representseither an uracil nucleotide, adenine nucleotide or cytosine nucleotide,V represents either an adenine nucleotide when S is a cytosinenucleotide or an uracil or cytosine nucleotide when S is an adeninenucleotide, Y₁ through Y₄ represent nucleotides that are respectivelycomplementary to W₁ through W₄, Z₁ through Z_(m) represent nucleotidesthat are respectively complementary to X₁ through X_(m), and m and n arethe same or different from each other and represent an integer from 1 to10.
 15. The process for cleaving the polyribonucleotide ζ in the formula(VI) according to claim 14, wherein L is an uracil nucleotide and V is acytosine nucleotide, wherein S is an adenine nucleotide and n is
 3. 16.A DNA that codes for the polyribonucleotide according to claim 12 or 13.17. A recombinant vector that includes the DNA according to claim 16.18. A host cell tranfected with the recombinant vector according toclaim
 17. 19. The polyribonucleotide according to claim 1, which isselected from the group consisting of ##STR28##
 20. Thepolyribonucleotide of claim 1, which is ##STR29##
 21. Thepolyribonucleotide of claim 1, which is ##STR30##
 22. Thepolyribonucleotide according to claim 1, which is ##STR31##